Glycosylation in Cellular Mechanisms of Health and Disease

Glycosylation produces an abundant, diverse, and highly regulated repertoire of cellular glycans that are frequently attached to proteins and lipids. The past decade of research on glycan function has revealed that the enzymes responsible for glycosylation—the glycosyltransferases and glycosidases—are essential in the development and physiology of living organisms. Glycans participate in many key biological processes including cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. This review discusses the increasingly sophisticated molecular mechanisms being discovered by which mammalian glycosylation governs physiology and contributes to disease. Glycosylation produces an abundant, diverse, and highly regulated repertoire of cellular glycans that are frequently attached to proteins and lipids. The past decade of research on glycan function has revealed that the enzymes responsible for glycosylation—the glycosyltransferases and glycosidases—are essential in the development and physiology of living organisms. Glycans participate in many key biological processes including cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. This review discusses the increasingly sophisticated molecular mechanisms being discovered by which mammalian glycosylation governs physiology and contributes to disease. Glycans are one of the four basic components of cells and may also be the most abundant and diverse of nature's biopolymers. Existing as covalent linkages of saccharides often attached to proteins and lipids, glycans constitute a significant amount of the mass and structural variation in biological systems. The field of glycobiology is focused upon understanding the structure, chemistry, biosynthesis, and biological function of glycans and their derivatives. Glycobiology has a long history that began with investigations of the basic constituents of cells and the nature of the polysaccharide carbohydrate component. Clinical applicability arose early with the discovery of the human blood groups, although evidence that these were glycan antigens came later (Landsteiner, 1931Landsteiner K. Individual differences in human blood.Science. 1931; 73: 405-411Crossref Scopus (43) Google Scholar). In addition, the antithrombotic glycan heparin is one of the most commonly used drugs (Linhardt, 1991Linhardt R.J. Heparin: an important drug enters its seventh decade.Chem. Ind. 1991; 2: 45-50Google Scholar, Shriver et al., 2004Shriver Z. Raguram S. Sasisekharan R. Glycomics: A pathway to a class if new and improved therapeutics.Nat. Rev. Drug Discov. 2004; 3: 863-873Crossref PubMed Scopus (179) Google Scholar), with current estimates of a billion doses prescribed annually. As glycobiology is increasingly interrelated with other disciplines, nomenclature and terminology within the field continue to evolve. The word glycan is now often used to encompass oligosaccharide, polysaccharide, and carbohydrate, as not all glycans are oligomers and the term carbohydrate can be confused with components of intermediary energy metabolism. More recently, in parallel with approaches to define genomics, proteomics, and lipidomics, the term "glycomics" has emerged, which refers to the study of the glycan structures that compose an organism's "glycome." The mammalian glycome repertoire is estimated to be between hundreds and thousands of glycan structures, and could be larger than the proteome. Although the diversity of glycan structures theoretically is vast, constraints are provided by the mechanisms of glycan synthesis and regulation. Mammalian glycans are formed by an endogenous portfolio of cellular enzymes and substrates that have been retained in an evolutionary investment encompassing millions of years and spanning 1%–2% of the genome. Vertebrates, and especially mammals, have evolved a highly complex glycan repertoire that is structurally distinct from that of invertebrates, lower eukaryotes, and prokaryotes. It is increasingly evident that the variation in glycomes among organisms is a molecular basis for interspecies recognition systems. Glycans of nonvertebrate organisms, for example, can modulate the development and activation of the mammalian immune system (Cobb and Kasper, 2005Cobb B.A. Kasper D.L. Coming of age: carbohydrates and immunity.Eur. J. Immunol. 2005; 35: 352-356Crossref PubMed Scopus (71) Google Scholar). Mammalian glycans are remarkably well conserved, but species-specific variations also exist, and these differences may be involved in the emergence of distinct traits including susceptibilities to infectious pathogens (Gagneux and Varki, 1999Gagneux P. Varki A. Evolutionary considerations in relating oligosaccharide diversity to biological function.Glycobiology. 1999; 9: 747-755Crossref PubMed Scopus (391) Google Scholar; also see the Essay by A. Varki, page 841 of this issue). Engineering new chemical modifications into glycans of living cells may improve the ability to detect glycan function and contribute to future diagnosis and treatment of disease (Prescher et al., 2004Prescher J.A. Dube D.H. Bertozzi C.R. Chemical remodeling of cell surfaces in living animals.Nature. 2004; 430: 873-877Crossref PubMed Scopus (493) Google Scholar; also see the Minireview by J. Prescher and C. Bertozzi, page 851 of this issue). Indeed, glycosylation defects in mice as well as humans and their links to disease have shown that the mammalian glycome contains a significant amount of biological information (Lowe and Marth, 2003Lowe J.B. Marth J.D. A genetic approach to mammalian glycan function.Annu. Rev. Biochem. 2003; 72: 673-691Crossref Scopus (496) Google Scholar, Freeze, 2006Freeze H.H. Genetic defects in the human glycome.Nat. Rev. Genet. 2006; 7: 537-551Crossref PubMed Scopus (349) Google Scholar). This review focuses upon the involvement of mammalian glycans in the molecular and cellular mechanisms that control health and disease. Nine monosaccharides are used in the enzymatic process of glycosylation in mammals. Conserved biosynthetic pathways provide all nine monosaccharides from sugars and precursors ubiquitously present in the diet. Except in cases of rare genetic defects, dietary intake of monosaccharides or mammalian glycans has not been rigorously established to have a beneficial effect on human health or in the treatment of disease. Biosynthetic pathways control the production and endogenous functions of different glycan structures. The structural diversity of the mammalian glycome is produced predominantly in the secretory pathway of the cell. Moreover, it is within the Golgi apparatus that glycans become increasingly oligomeric and branched as they transit through this latter portion of the secretory system bound mostly for the cell surface and extracellular compartments. Glycosylation produces different types of glycans (or glycoconjugates) that are typically attached to cellular proteins and lipids (Figure 1). Protein glycosylation encompasses N-glycans, O-glycans, and glycosaminoglycans (frequently termed proteoglycans). N-glycans are linked to asparagine residues of proteins, specifically a subset residing in the Asn-X-Ser/Thr motif, whereas O-glycans are attached to a subset of serines and threonines (Schachter, 2000Schachter H. The joys of HexNAc. The synthesis and function of N- and O-glycan branches.Glycoconj. J. 2000; 17: 465-483Crossref PubMed Scopus (121) Google Scholar, Yan and Lennarz, 2005Yan A. Lennarz W.J. Unraveling the mechanism of protein N-glycosylation.J. Biol. Chem. 2005; 280: 3121-3124Crossref PubMed Scopus (146) Google Scholar). Although glycosaminoglycans are also linked to serine and threonine, they are linear, are produced by different biosynthetic pathways, and are often highly sulfated (Esko and Selleck, 2002Esko J.D. Selleck S.B. Order out of chaos: assembly of ligand binding sites in heparan sulfate.Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1115) Google Scholar). Lipid glycosylation in the secretory pathway is also a prevalent modification and creates glycolipids (glycosphingolipids) that include the sialic acid-bearing gangliosides (Maccioni et al., 2002Maccioni H.J. Giraudo C.G. Danniotti J.L. Understanding the stepwise synthesis of glycolipids.Neurochem. Res. 2002; 27: 629-636Crossref PubMed Scopus (42) Google Scholar). Glycosylphosphatidylinositol (GPI)-linked proteins share a common membrane-bound glycolipid linkage structure that is attached to various proteins (Kinoshita et al., 1997Kinoshita T. Ohishi K. Takeda J. GPI-anchor synthesis in mammalian cells: genes, their products, and a deficiency.J. Biochem. (Tokyo). 1997; 122: 251-257Crossref PubMed Scopus (115) Google Scholar). Hyaluronan is a unique glycan type unattached to either proteins or lipids that is secreted into extracellular compartments (Weigel et al., 1997Weigel P.H. Hascall V.C. Tammi M. Hyaluronan synthases.J. Biol. Chem. 1997; 272: 13997-14000Crossref PubMed Scopus (575) Google Scholar). Less common types of protein glycosylation also occur, for example, on lysine, tryptophan, and tyrosine residues of specific proteins, such as glycogen, which was the first identified glycoprotein. In addition, although technically not glycosylation, acetyltransferase and sulfotransferase enzymes residing in the secretory pathway frequently attach acetyl and sulfate groups to selected saccharides residing on some oligosaccharide chains and can thereby modulate glycan structure and function (Klein and Roussel, 1998Klein A. Roussel P. O-acetylation of sialic acids.Biochimie. 1998; 80: 49-57Crossref PubMed Scopus (79) Google Scholar, Fukuda et al., 2001Fukuda M. Hiraoka N. Akama T.O. Fukuda M.N. Carbohydrate-modifying sulfotransferases: structure, function, and pathophysiology.J. Biol. Chem. 2001; 276: 47747-47750Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Some forms of glycosylation occur outside of the secretory pathway. Among most eukaryotic organisms, N-acetylglucosamine has been found linked to serine and threonine residues (O-GlcNAc) on many cytoplasmic and nuclear proteins (Hart, 1997Hart G.W. Dynamic O-linked glycosylation of nuclear and cytoskeletal proteins.Annu. Rev. Biochem. 1997; 66: 315-335Crossref PubMed Scopus (425) Google Scholar). Similar to protein phosphorylation, GlcNAcylation is an enzymatic modification that typically has a shorter half-life than that of the attached proteins. This reflects the presence of a regulated cytoplasmic N-acetylglucosaminidase, which removes O-GlcNAc, leaving the serine or threonine residue subsequently available for another round of GlcNAcylation or sometimes phosphorylation. O-GlcNAc is a highly regulated posttranslational modification required for the viability of many mammalian cell types perhaps by acting as a nutrient sensor, preventing protein phosphorylation, or regulating protein turnover (Zhang et al., 2003Zhang F. Su K. Yang X. Bowe D.B. Paterson A.J. Kudlow J.E. O-GlcNAc modification is an endogenous inhibitor of the proteasome.Cell. 2003; 115: 715-725Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, O'Donnell et al., 2004O'Donnell N. Zachara N.E. Hart G.W. Marth J.D. Ogt-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability.Mol. Cell. Biol. 2004; 24: 1680-1690Crossref PubMed Scopus (287) Google Scholar, Zachara and Hart, 2004Zachara N.E. Hart G.W. O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress.Biochim. Biophys. Acta. 2004; 1673: 13-28Crossref PubMed Scopus (291) Google Scholar). It is useful to distinguish secretory and cytoplasmic glycosylation from glucuronidation, the latter being an enzymatic process linking a single glucuronic acid to bile salts and xenobiotics (molecules that are foreign to cells) (Tukey and Strassburg, 2000Tukey R.H. Strassburg C.P. Human UDP-glucuronosyltransferases: metabolism, expression, and disease.Annu. Rev. Pharmacol. Toxicol. 2000; 40: 581-616Crossref PubMed Scopus (1193) Google Scholar). In contrast to glycosylation, glycation refers to the covalent linkage of saccharides such as glucose to proteins by a nonenzymatic and irreversible process that is elevated in various diseases and may be a factor in the pathology of aging (Suji and Sivakami, 2004Suji G. Sivakami S. Glucose, glycation, and aging.Biogerontology. 2004; 5: 365-373Crossref PubMed Scopus (66) Google Scholar). Glycans are constructed in an ordered sequential manner involving the distinct substrate specificities of glycosyltransferase and glycosidase enzymes. Glycosyltransferases synthesize glycan chains, whereas glycosidases hydrolyze specific glycan linkages. Although glycosyltransferases are the anabolic component of glycosylation, both types of enzymes collaborate to determine the structural outcome in pathways of glycan biosynthesis. Such properties are exemplified by mammalian N-glycan biosynthesis (Kornfeld and Kornfeld, 1985Kornfeld S. Kornfeld R. Assembly of asparagine-linked oligosaccharides.Annu. Rev. Biochem. 1985; 54: 631-644Crossref PubMed Scopus (3639) Google Scholar). Similarly, the biosynthetic pathways for the production of O-glycans, glycosaminoglycans, and glycolipids are comprised of single enzymatic steps that typically rely upon glycan structures produced by the previous enzyme to produce the substrate for the next (Schachter, 2000Schachter H. The joys of HexNAc. The synthesis and function of N- and O-glycan branches.Glycoconj. J. 2000; 17: 465-483Crossref PubMed Scopus (121) Google Scholar, Maccioni et al., 2002Maccioni H.J. Giraudo C.G. Danniotti J.L. Understanding the stepwise synthesis of glycolipids.Neurochem. Res. 2002; 27: 629-636Crossref PubMed Scopus (42) Google Scholar, Esko and Selleck, 2002Esko J.D. Selleck S.B. Order out of chaos: assembly of ligand binding sites in heparan sulfate.Annu. Rev. Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1115) Google Scholar). Although a one enzyme/one saccharide linkage paradigm applies to almost all biosynthetic steps, numerous glycosyltransferase isozymes exist, and these underlie the breadth of glycan participation among different cell types and physiological processes. Glycosylation in the secretory pathway is a dynamic process with multiple mechanisms that alter glycosyltransferase and glycosidase expression and structure, as well as their accessibility to substrates. Thus, in concert with protein and lipid turnover, glycosylation can regulate glycan variation (Figure 2). Structural variations in the glycan repertoire at the cell surface produce numerous biomarkers, some of which correlate with differentiation, cell activation, and disease. For example, elevated levels of truncated O-glycans (known as T antigens) can be prognostic for reduced survival of patients with certain types of cancer (Hakomori, 2002Hakomori S. Glycosylation defining cancer malignancy: new wine in an old bottle.Proc. Natl. Acad. Sci. USA. 2002; 99: 10231-10233Crossref PubMed Scopus (732) Google Scholar, Kobata and Amano, 2005Kobata A. Amano J. Latered glycosylatiohn of proteins produced by malignant cells, and applications in the diagnosis and immunotherapy of tumors.Immunol. Cell Biol. 2005; 83: 429-439Crossref PubMed Scopus (208) Google Scholar). Gene transcription has a major impact on glycan formation, which is reflected by the cell-type-specific and developmentally modulated RNA expression profiles observed among many glycosyltransferases and glycosidases. Microarray approaches that detect the transcript levels of enzymes involved in constructing the glycome will be increasingly useful in categorizing these changes and perhaps provide predictive information on cellular glycan expression patterns (Comelli et al., 2006Comelli E.M. Head S.R. Gilmartin T. Whisenant T. Haslam S.M. North S.J. Wong N.K. Kudo T. Narimatsu H. Esko J.D. et al.A focused microarray approach to functional glycomics: transcriptional regulation of the glycome.Glycobiology. 2006; 16: 117-131Crossref PubMed Scopus (131) Google Scholar). Transcriptional regulation of RNA abundance occurs among glycosyltransferase genes encoding, for example, GlcNAcT-V and Core 2 GlcNAcT-I. The GlcNAcT-V gene promoter bears Ets transcription factor binding elements that induce transcription in response to signals emanating from several key regulators of cell proliferation, including the Her-2/Neu oncogene. Core 2 GlcNAcT-I is induced by the T-bet transcription factor in T helper type 1 lymphocytes (Chen et al., 1998Chen L. Zhang W. Fregien N. Pierce M. The her-2/neu oncogene stimulates the transcription of N-acetylglucosaminyltransferase V and expression of its cell surface oligosaccharide products.Oncogene. 1998; 17: 2087-2093Crossref PubMed Scopus (82) Google Scholar, Underhill et al., 2005Underhill G.H. Zisoulis D.G. Kolli K.P. Ellies L.G. Marth J.D. Kansas G.S. A crucial role for T-bet in selectin ligand expression in T helper 1 (Th1) cells.Blood. 2005; 106: 3867-3873Crossref PubMed Scopus (43) Google Scholar). Although multiple transcriptional networks regulate glycosyltransferase and glycosidase gene expression, the effect of this regulation on cellular processes remains largely unknown. The expression of mammalian glycans is regulated at both a posttranscriptional and posttranslational level. Currently, this does not appear to involve modulation of enzymatic activity, as glycosyltransferases and glycosidases are constitutively active. Nevertheless, some must be properly glycosylated themselves to be active, which suggests a possible mechanism of catalytic regulation in vivo. Mechanisms altering the intracellular location of glycosyltransferases and glycosidases can be an effective means of regulating glycan formation by controlling access to acceptor substrates. Major changes in the glycome are induced by the loss of some chaperones and multiprotein complexes that alter glycosyltransferase trafficking between the endoplasmic reticulum and Golgi (Wu et al., 2004Wu X. Steet R.A. Bohorov O. 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A potential means by which glycosyltransferases and glycosidases may be regulated is through phosphorylation of their cytoplasmic tails, which might modulate intermolecular interactions leading to differential substrate access and intracellular trafficking. There is also evidence of competition among glycosyltransferases in vivo for substrates in the secretory pathway, which can modify glycan formation. Some glycosyltransferases that generate different saccharide linkages have distinct specificities for nucleotide sugar donors but the same acceptor substrate specificity; whereas others bear identical donor specificity but act on different acceptor substrates. Glycosyltransferases of the former type can be mutually exclusive in the assembly line of glycan formation; whichever enzyme modifies the substrate first can thereby redirect the synthetic pathway and alter the structural outcome. This was observed by the in vivo blockade of Core 2 GlcNAcT function due to endogenous expression of ST3Gal-I in T cells. Both glycosyltransferases can act on the same acceptor substrate, and loss of ST3Gal-I elevated Core 2 O-glycan synthesis without a change in Core 2 GlcNAcT enzyme activity (Priatel et al., 2000Priatel J.J. Chui D. Hiraoka N. Simmons C.J. Richardson K.B. Page D.M. Fukuda M. Varki N.M. Marth J.D. The ST3Gal-I sialyltransferase controls CD8+ T lymphocyte homeostasis by modulating O-glycan biosynthesis.Immunity. 2000; 12: 273-283Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Disengagement of glycosyltransferases and glycosidases from their membrane-anchored locations can occur by proteolysis and would be expected to abolish their activities in glycan formation, although evidence of this potential form of glycan regulation currently awaits further experimentation. The enzymes of mammalian glycosylation are predominantly type 2 transmembrane glycoproteins that contain a luminal catalytic domain linked to a luminal membrane-proximal "stem" domain. Cleavage by secretory proteases within the stem domain results in secretion of a catalytic domain fragment. This fragment can be found in most body fluids and can be induced, for example, in response to inflammation (McCaffrey and Jamieson, 1993McCaffrey G. Jamieson J.C. Evidence for the role of a cathepsin D-like activity in the release of Gal beta 1-4GlcNAc alpha 2-6sialyltransferase from rat and mouse liver in whole-cell systems.Comp. Biochem. Physiol. B. 1993; 104: 91-94Crossref PubMed Scopus (21) Google Scholar). The purpose of this proteolysis is unknown, and the range of enzymes affected is unclear. Although such glycosyltransferase fragments retain enzymatic activity, and hence their ability to bind to available acceptor substrates, they are not likely to be catalyzing glycan formation among extracellular compartments, as the concentration of nucleotide sugar donors outside of the cell's secretory pathway is far below enzyme substrate binding affinities. The hydrolysis of glycans on mammalian glycoproteins and glycolipids is associated with their degradation in lysosomes. However, endogenous mechanisms that cleave glycans at the cell surface may exist. Hydrolysis of mammalian cell-surface glycans is in fact a feature of some pathogen infection strategies such as sialic acid binding and cleavage by influenza virus (Gagneux and Varki, 1999Gagneux P. Varki A. Evolutionary considerations in relating oligosaccharide diversity to biological function.Glycobiology. 1999; 9: 747-755Crossref PubMed Scopus (391) Google Scholar; also see the Minireview by L. Comstock and D. Kasper, page 847 of this issue). Proteolysis and trafficking to the cell surface would place mammalian glycosidase enzymes in the region of the cell-surface glycocalyx, where some glycans might be hydrolyzed. At least one mammalian glycosidase that cleaves sialic acids from glycans is a transmembrane protein that reaches the plasma membrane (Wang et al., 2004Wang P. Zhang J. Bian H. Wu P. Kuvelkar R. Kung T.T. Crawley Y. Egan R.W. Billah M.M. Induction of lysosomal and plasma membrane-bound sialidases in human T-cells via T-cell receptor.Biochem. J. 2004; 380: 425-433Crossref PubMed Scopus (41) Google Scholar). Although examples of cell-surface glycoprotein alterations consistent with removal of specific glycan linkages have been described—such as the highly reproducible reduction in some sialic acid linkages following immune activation of mammalian lymphocytes—this may be explained by endocytosis and turnover in which newly synthesized glycoproteins bear different glycans due to modulation of glycosyltransferase or glycosidase function. The biosynthesis and availability of nucleotide sugar donor substrates can exert broad control over mammalian glycan formation. Blockade of donor biosynthesis or functional loss of donor-specific transporters normally residing the endoplasmic reticulum and Golgi membranes can abolish cellular glycans that contain, for example, fucose or sialic acid linkages (Lubke et al., 2001Lubke T. Marquardt T. Etzioni A. Harmann E. von Figura K. Korner C. Complementation cloning identifies CDG-IIc, a new type of congenital disorders of glycosylation, as a GDP-fucose transporter deficiency.Nat. Genet. 2001; 28: 73-76Crossref PubMed Google Scholar, Smith et al., 2002Smith P.L. Myers J.T. Rogers C.E. Zhou L. Petryniak B. Becker D.J. Homeister J.W. Lowe J.B. Conditional control of selectin ligand expression and global fucosylation events in mice with a targeted mutation at the FX locus.J. Cell Biol. 2002; 158: 801-815Crossref PubMed Scopus (121) Google Scholar, Schwarzkopf et al., 2002Schwarzkopf M. Knobeloch K.P. Rohde E. Hinderlich S. Wiechens N. Lucka L. Horak I. Reutter W. Horstkorte R. Sialylation is essential for early development in mice.Proc. Natl. Acad. Sci. USA. 2002; 99: 5267-5270Crossref PubMed Scopus (248) Google Scholar). In contrast, glucosamine supplementation to the hexosamine biosynthetic pathway can elevate synthesis of some donor substrates and increase production of various glycans in mammalian cells (Zachara and Hart, 2004Zachara N.E. Hart G.W. O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress.Biochim. Biophys. Acta. 2004; 1673: 13-28Crossref PubMed Scopus (291) Google Scholar, Lau et al., 2005Lau K. Partridge E.A. Cheung P. Dennis J.W. Hexosamine, N-glycans, and cytokine signaling-a regulatory network.Glycobiology. 2005; 15: 1196Google Scholar). Precisely how this occurs may reflect multiple factors including increased catalysis and changes in gene expression. The impact of such augmented glycosylation upon mammalian physiology is not yet known, although this matter is worthy of careful investigation. With a number of regulatory mechanisms available, several and perhaps all of those discussed above are involved in modulating mammalian glycan expression. Few biological roles for mammalian glycosylation had been established even a decade ago. The rapid pace of discovery since then reflects the application of genetic tools and approaches to expand upon the existing foundation of enzymatic, biochemical, and structural knowledge. Glycosylation, like phosphorylation, produces numerous structural modifications, each of which may be capable of signaling. Likewise, absence of a single kinase or glycosyltransferase affects the modification of multiple proteins and lipids. In studies of phosphorylation, this is commonly interpreted as disruption of a signal transduction cascade. In glycosylation, the specificity of most glycosyltransferases and glycosidases for substrates is defined by glycan structure instead of protein and lipid determinants. Therefore, single enzymes can glycosylate multiple, seemingly unrelated, proteins and lipids. How then does glycan formation achieve a high level of specificity in cellular function? The answer may come from combining knowledge of glycan synthesis and regulation with the phenotypes observed in intact organisms bearing defects in glycan formation. Cultured cells bearing various enzymatic defects in the pathways of glycosylation typically lack significant phenotypes, yet a high degree of evolutionary conservation is typical among mammalian glycosyltransferase and glycosidase orthologs (Amado et al., 1999Amado M. Almeida R. Schwientek T. Clausen H. Identification and characterization of large galactosyltranserase gene families: galactosyltransferases for all functions.Biochim. Biophys. Acta. 1999; 1473: 35-53Crossref PubMed Scopus (245) Google Scholar, Kikuchi and Narimatsu, 2006Kikuchi N. Narimatsu H. Bioinformatics for comprehensive finding and analysis of glycosyltransferases.Biochim. 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USA. 1994; 91: 728-732Crossref PubMed Scopus (337) Google Scholar, Metzler et al., 1994Metzler M. Gertz A. Sarkar M. Schachter H. Schrader J.W. Marth J.D. Complex asparagine-linked oligosaccharides are required for morphogenic events during post-implantation development.EMBO J. 1994; 13: 2056-2065Crossref PubMed Google Scholar). Since these findings, dozens of mouse lines have been created bearing germline defects in specific steps of the various glycosylation pathways. Remarkably, most of these inherited glycan deficiencies result in discrete phenotypes that reflect the dysfunction of specific cell types and diverse biological systems (Lowe and Marth, 2003Lowe J.B. Marth J.D. A genetic approach to mammalian glycan function.Annu. Rev. Biochem. 2003; 72: 673-691Crossref Scopus (496) Google Scholar). Glycans possess distinct structural elements that govern interactions with other molecules. Glycans can promote or inhibit intra- and intermolecular binding that includes both homotypic and heterotypic interactions (Figure 3). Furthermore, mammalian glycans can be so substantial in size and frequency of attachment that they contribute the majority of mass and charge comprising some glycoproteins and glycolipids. For example, the neural cell adhesion molecule NCAM has a uniquely large negatively charged and devel